This invention relates to a method for identifying and in vitro sorting undifferentiated stem cells derived from migrating cranial neural crest (CNC) or from paraxial mesoderm of the first branchial arch. This invention also relates to the isolation, culture, proliferation, preservation and therapeutic use of these cells and their progeny.
Development of the mammalian nervous system (NS) begins in the early stages of fetal development and continues into the early post-natal period. Neural stem cells give rise to daughter stem cells and to neuroblasts and glioblasts. The mature mammalian nervous system is composed of neuronal cells (neurons and their axons), and of glial cells (astrocytes and oligodendrocytes). Neurons, the functional unit of the NS, are responsible for forming axonal connections with other neurons and with other functional end units (end organ sensory receptors, motor end plates, etc.)—they are the communicating cells of the NS. Astrocytes and oligodendrocytes provide a supportive role for optimal neuronal function.
Cells derived from neural tube give rise to the neurons and glia of the central nervous system (CNS) while cells derived from neural crest give rise to the peripheral nervous system (PNS). The neural crest also secretes nerve growth factor (NGF) which stimulates the development of neuronal axons throughout the NS.
Neurogenesis occurs primarily in two waves—a pre-natal wave during which most of the neurons are formed and an early post-natal wave during which most of the astrocytes and oligodendrocytes develop. The formation of neurons occurs in the fetal period and is completed by the early post-natal period. By the late post-natal period, the CNS has its full complement of nerve cells. Unlike other tissues, differentiated cells of the adult mammalian CNS demonstrate little ability to generate new nerve cells. While it is believed there is a slow or limited turnover of astrocytes and that progenitor cells for oligodendrocytes exist, the regeneration of new neurons is limited, particularly in adult primates. This limited ability of the CNS to produce new neurons is thought to be an advantage for long-term memory retention and for learned motor/sensory reflexes but it is a distinct disadvantage when the need to replace lost neurons or glial cells arises due to traumatic injury, neurological disease, or degenerative changes.
Growth-factor responsive cells from pre-natal and post-natal CNS exhibiting neural stem cell characteristics in vitro were isolated in the early 1990's (Reynolds, B. A. et. al., “Generations of neurons and astrocytes from isolated cells of the adult mammalian central nervous system,” Science 255:1707-1710 (1992)). The location of a small quantity of neural stem cells capable of differentiating into neurons and glia has been identified in the post-natal brain (Lois, S. et. al., “Proliferating subventricular zone cells in the adult mammalian forebrain can differentiate into neurons and glia,” Proc. Natl. Acad. Sci. USA, 90:2074-2077 (1993); Morshead, C. M. et. al., “Neural stem cells in the adult mammalian forebrain: a relatively quiescent subpopulation of subependymal cells,” Neuron, 13(5): 1071-1082 (1994)). These studies have opened the door to an emerging area of neurobiological research, namely repair based on the generation of new cells within the NS.
Development of the mammalian dentition, dentinogenesis, begins in the early stages of fetal development and continues well beyond the early post-natal period. Tooth formation reflects a complex sequence of epithelial-mesenchymal interactions occurring between enamel organ epithelium (EOE) cells, derived from ectoderm, and cranial neural crest (CNC) and non-cranial neural crest (non-CNC), derived from mesoderm located within the first branchial arch. In addition to participating in tooth development, migrating cranial neural crest (CNC) cells also contribute to the central and peripheral nervous systems through development of the cranial nerves, the eye (and its associated muscles), and other structures.
A two-component genetic marking system has been utilized during craniofacial development to systematically analyze the migration and differentiation of CNC-derived ectomesenchyme from early embryogenesis onward. See, Chai, Y. et. al., “Fate of the mammalian cranial neural crest during tooth and mandibular morphogenesis,” Development 127: 1671-1679 (2000). As described in Chai, at the initiation of tooth development (dental lamina stage, around 6-8 weeks in utero), the underlying mesenchyme within the maxillary and mandibular processes of the first branchial arch is composed almost entirely of migrating CNC-derived mesenchyme. As each individual tooth germ progresses from toothbud to cap stage, its CNC-derived mesenchyme begins to concentrate near remnants of the enamel organ epithelium (EOE), the structure responsible for enamel formation, while the remaining mesenchyme within the dental sac and dental papilla begins to demonstrate a mixture of CNC-derived mesenchyme and non-CNC-derived, or paraxial mesenchyme. Therefore, mesenchyme that will eventually form the mature dental pulp is populated by two lineages: CNC-derived mesenchyme and non-CNC-derived paraxial mesenchyme. CNC-derived mesenchyme concentrates in a circle of pulp tissue at the periphery of each developing tooth root adjacent to the epithelial root sheath and epithelial diaphragm, known as the “dental papillary ring” or “dental papillary annulus.”. This area of the of the developing pulp, adjacent to ectodermal structures derived from enamel organ epithelium (EOE), attracts a higher concentration of CNC-derived undifferentiated cells while the central core of the developing pulp is populated with a higher concentration of paraxial (non-CNC-derived) mesenchyme.
Until apical closure of the developing tooth root occurs, developing dental pulp is properly termed “dental papilla,” the mesenchymal structure which gives rise to the dental pulp and to the tooth's dentin. Only following apical closure is the term “dental pulp” properly applied. Therefore, dental pulp exists only after it is fully enclosed by dentin, also derived from dental papilla. For the proposes of this invention, “dental papillary annulus” or “dental papillary ring” refers to that portion of the developing pulp tissue at the periphery of each developing tooth root that is adjacent to the epithelial root sheath and epithelial diaphragm regardless of whether the term dental pulp or dental papilla is used to refer to the developing pulpal tissue.
Nervous system disorders include neurodegenerative diseases, injuries, tumors, and a large number of central nervous system (CNS) dysfunctions. While not limited to the elderly, neurodegenerative diseases have gained increasing attention because of their occurrence in an expanding elderly population, which is at greater risk. Cerebral Palsy, Multiple Sclerosis, Amyotrophic Lateralizing Sclerosis, Epilepsy, and diseases such as Alzheimer's disease, Parkinson's disease, Huntington's disease, have all been linked to the degeneration of neural cells in particular locations of the central nervous system, leading to the inability of these regions to function normally. CNS injuries and tumors often result in the loss of neural cells, inappropriate function of the affected region, and a subsequent constellation of sensory, motor or behavioral abnormalities. Other CNS dysfunctions which affect a large number of people are not characterized by a loss of neural cells but by the abnormal function of existing cells due to inappropriate neuronal function or to the abnormal synthesis, release or processing of neurotransmitters resulting in disorders such as autism, depression, neurosis and psychosis.
In the case of injuries and tumors, treatment for CNS disorders has primarily been interventional. In the case of neurodegenerative diseases and CNS dysfunction, treatment has primarily been pharmacologic with administration of agents designed to normalize function. Pharmacological therapy is limited by certain inherent difficulties including transportation of the drug across the blood-brain barrier, acquired drug tolerance, and drug side-effects. It would be advantageous to have a reliable, post-natal source of neural cells available at least for the study of CNS dysfunction, and more preferably for drug development and screening, as well as for regenerative medicine and tissue engineering.